Chondroitin, a type of glycosaminoglycan, is a glycan (sugar chain) having a basic backbone of a repeating disaccharide structure [→4)-β-GlcUA-(1→3)-β-GalNAc-(1→] consisting of glucuronic acid (GlcUA) and N-acetylgalactosamine (GalNAc). In the body of animals, chondroitin typically occurs as chondroitin sulfate that is sulfated at some of the hydroxyl groups of the constituent sugars. For example, chondroitin that is sulfated at the hydroxyl group in position 4 of the GalNAc residues (chondroitin-4-sulfate) is called chondroitin sulfate A, and chondroitin that is sulfated at the hydroxyl group in position 6 of the GalNAc residues (chondroitin-6-sulfate) is called chondroitin sulfate C.
Patent Document 1 describes a protein having covalently linked chondroitin. The document also describes that the protein has improved stability against degradation in vivo compared to unmodified one. However, in the document, the term “chondroitin” is intended to mean “chondroitin sulfate”, such as chondroitin-4-sulfate and chondroitin-6-sulfate (see the top left column in page 4 of the document).
Patent Document 2 describes a physiologically active human thrombomodulin polypeptide, which is an isolated polypeptide having a glycan comprising chondroitin and/or chondroitin sulfate in the peptide chain (type-II thrombomodulin polypeptide). The document also describes that the polypeptide has higher activity than a polypeptide substantially having no glycan comprising chondroitin and/or chondroitin sulfate (type-I thrombomodulin polypeptide).
Patent Document 3 describes a protein modified with a glycosaminoglycan. The document also describes that the protein is more stable in vivo and capable of more sustainably developing physiological activity, than unmodified one. Moreover, Patent Document 4 describes a covalent conjugate comprising of a glycosaminoglycan linked to a protein by a covalent linkage. The document also describes that pharmacokinetic properties are improved by conjugation of a glycosaminoglycan. Though Patent Documents 3 and 4 refer to chondroitin, chondroitin is merely exemplified as a material that falls in the same category as many other glycosaminoglycans (i.e. colominic acid, hyaluronic acid, chondroitin sulfate, teichuronic acid, dermatan sulfate, heparin, heparan sulfate, keratan sulfate, keratan polysulfate, and derivatives thereof). In fact, these documents do not disclose or suggest whether or not protein stability depends on the type of glycosaminoglycan conjugated to the protein.
Furthermore, the foregoing documents do not disclose or suggest using chondroitin produced by a microorganism having chondroitin-producing capability or chondroitin synthesized with a chondroitin synthase.
In nature, chondroitin is to be present in nematodes, eye cornea of cow, skin of Japanese flying squid (Todarodes pacificus) and common octopus (Octopus vulgaris), and the like. However, since natural chondroitin are scarce, it is impractical to produce chondroitin from these sources in industrial scale. Accordingly, chondroitin is typically produced by desulfation of chondroitin sulfate, and such chondroitin is generally used as chondroitin.
In recent years, there have been reports of chondroitin production by using a microorganism. Patent Documents 5 to 7 describe bacteria of Escherichia having chondroitin-producing capability and chondroitin produced by the bacteria. Patent Documents 8 and 9 disclose methods for synthesizing chondroitin in vitro with a chondroitin synthase. However, these documents do not disclose or suggest using chondroitin produced by microorganisms such as bacteria or chondroitin synthesized with a chondroitin synthase for increasing protein retention in blood.
Moreover, Patent Documents 1 to 9 do not disclose or suggest that retention of a glycosaminoglycan itself in blood varies with the type or the origin of the glycosaminoglycan. These documents also do not disclose or suggest that chondroitin produced by a microorganism or chondroitin synthesized with an enzyme have much longer retention in blood than other glycosaminoglycans. Furthermore, Patent Documents 1 to 9 do not disclose or suggest that a complex consisting of a protein conjugated with chondroitin produced by a microorganism or chondroitin synthesized with an enzyme have much longer retention in blood than a complex consisting of a protein conjugated with chondroitin obtained through desulfation of chondroitin sulfate or other glycosaminoglycans.